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Microglia in Health and Disease

Richard M. Ransohoff1 and Joseph El Khoury2

1Neuroinflammation Research Center, Department of Neurosciences, Cleveland Clinic Lerner ResearchInstitute, Cleveland, Ohio 44195

2Division of Infectious Diseases and Center for Immunology and Inflammatory Diseases, MassachusettsGeneral Hospital, Harvard Medical School, Charlestown, Massachusetts 02129

Correspondence: [email protected]; [email protected]

Microglia, the major myeloid cells of the central nervous system (CNS) are implicated inphysiologic processes and in the pathogenesis of several CNS disorders. Since their initialdescription early in the 20th century, our ability to identify and isolate microglia has sig-nificantly improved and new research is providing insight into the functions of these cells insickness and in health. Here, we review recent advances in our understanding of the role ofmicroglia in physiological and pathological processes of the CNS with a focus on multiplesclerosis and Alzheimer’s disease. Because of the prominent roles CX3CR1 and its ligandfractalkine played in bringing about these advances, we discuss the physiological andpathological roles of microglia as viewed from the CX3CR1–fractalkine perspective, pro-viding a unique viewpoint. Based on the most recent studies of molecular profiling of microg-lia, we also propose a molecular and functional definition of microglia that incorporates theproperties attributed to these cells in recent years.

A BRIEF HISTORY OF MICROGLIA

Microglia, the major myeloid cells of the cen-tral nervous system (CNS), have long been

implicated in physiologic and pathologic pro-cesses. The 20th century brought several majorbreakthroughs in our ability to identify and iso-late these cells. In the past decade, new researchhas emerged providing evidence and insightinto the functions of these cells in sickness andin health.

The first breakthroughs in research on mi-crogliawere presented in a series of publications,nearly a century ago, by Pıo del Rıo-Hortega.In an article published in 1918, del Rıo-Hortegadescribed a method for staining microglia andwas therefore able to distinguish these cells from

other neighboring cells of the neural milieu (delRıo-Hortega 1918). In a subsequent article pub-lished the following year, del Rıo-Hortega de-scribed microglia as the “third element” in theCNS, he also emphasized their phagocytic ca-pacity (del Rıo-Hortega 1919). Additional workperformed by del Rıo-Hortega indirectly de-scribed microglial plasticity and discussedthe ability of these cells to change in responseto stimuli (del Rıo-Hortega 1920). He furtherwent on to describe the regional distributionof microglia and their heterogeneity (del Rıo-Hortega 1921). Since then, scientists investigat-ing microglia using progressively sophisticatedtechnologies have largely confirmed and builton del Rıo-Hortega’s initial observations.

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After del Rıo-Hortega’s initial findings, ittook another 50 years for the second wave ofbreakthroughs in microglia research to occur.Development of antibodies to various innateimmune cell proteins identified markers for mi-croglia expressed in situ such as F4/80 (Humeet al. 1983), Fc receptors, and complement re-ceptor 3 (Perry et al. 1985). Subsequently, Giu-lian and Baker (1986) described a novel methodto isolate and culture microglia from neonatalrodent brains. The ability to culture these cellsled to a large number of in vitro investigations todefine their functions. Cultured microglia werefound to be capable of phagocytosis and lyso-zyme production (Zucker-Franklin et al. 1987),and respond to purinergic stimulation (Walzet al. 1993) and to b-amyloid (Meda et al.1995; El Khoury et al. 1996). This approachalso led to identification of novel markers forthese cells such as Iba1 (Imai et al. 1996).

The availability of new markers for microg-lia provided tools to begin to investigate the roleof these cells in various CNS disorders. Presenceof these cells in senile plaques in Alzheimer’sdisease (AD) was confirmed using specificmonoclonal antibodies to a1 chymotrypsin, ly-sozyme (Rozemuller et al. 1986), and HLA-DR(McGeer et al. 1987). Microglia-expressingMHC II antigens were also identified in multiplesclerosis (MS) brains (Cuzner et al. 1988).

The third era of breakthroughs in mi-croglia research started with the generation ofa transgenic knockin mouse expressing greenfluorescent protein (GFP) driven by the pro-moter for the chemokine receptor CX3CR1(Jung et al. 2000). Because CX3CR1 is exclusive-ly expressed on microglia in the CNS, GFP wasexpressed only in these cells, allowing investi-gators to visualize microglia without the needfor immunohistochemistry or special staining.This mouse line ushered in a new line of inves-tigations exploring the roles of microglia in var-ious physiological and pathological processes.

The ability to visualize microglia withoutthe need for immunolabeling and the devel-opment of novel two-photon in vivo imagingmethodologies provided unprecedented viewsinto the role of these cells in vivo. In a landmarkpublication, Nimmerjahn et al. (2005) showed

that the morphologically “resting” microgliaare highly active, continually surveying theirmicroenvironment with extremely motile pro-cesses and protrusions; they also showed thatfocal injury to the brain provoked immediateand focal activation of microglia, switchingtheir behavior from patrolling to shielding ofthe injured site. At the same time, Davalos andcolleagues (2005) reported identical findingsand showed further that the rapid process–extension response to laser lesion was mediatedby ATP binding to the purinergic receptorP2ry12 (Haynes et al. 2006). Additional stud-ies, discussed in the following pages, used theCX3CR1 GFP mice (or improved transgenicversions of these mice) and two-photon invivo imaging to further explore the role of mi-croglia in synaptic pruning and remodeling,and in various disease models including ex-perimental autoimmune encephalitis (EAE),a model of MS, mouse models of amyloid de-position and tauopathies in AD, and models ofischemic injury.

The fourth era of breakthroughs in microg-lia research began when methods to isolate mi-croglia from adult mice were developed (Hick-man et al. 2008), leading to several landmarkfindings in 2013. With the increased recogni-tion of the important roles microglia couldplay in physiological and pathological process-es, and because of the multiple similarities anddifferences between these cells and other mono-nuclear phagocytes, it became crucial to de-velop a more accurate definition of microgliathat distinguishes them from other cells in theCNS as well as other mononuclear phagocytessuch as monocytes and macrophages. Over thepast year, using methodologies such as directRNA sequencing (RNASeq) (Chiu et al. 2013;Hickman et al. 2013), and microarrays (Beutneret al. 2013; Butovsky et al. 2014), four groupsindependently identified expression signaturesof microglia that help define these cells and de-termine their unique transcriptional character-istics.

One further notable advance in under-standing microglia came gradually over de-cades, so cannot conveniently be summarizedin a single breakthrough. Microglial ontogeny

R.M. Ransohoff and J. El Khoury

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was implied by del Rıo-Hortega as being mes-enchymal. However, unequivocal evidence forthis proposal was not forthcoming until 1996,when Maki and coworkers (McKercher et al.1996) showed that mice lacking transcriptionfactor PU.1 were devoid of microglia. In thesame time frame, Alliot and colleagues (1991,1999) conducted a series of descriptive studiesand proposed that microglia originate in theyolk sac, enter brain rudiment after E8 in rodentsand establish the adult microglial populationthrough vigorous in situ proliferation. Ajamiand colleagues followed in 2007, by showingthat microglia self-renew in the CNS without ablood-monocyte contribution. In 2010, Gin-houx and colleagues (2010) performed a defin-itive fate mapping confirming Pessac’s hypoth-esis. Two years later, Geissmann (Schulz et al.2012) showed the c-myb independence of mi-croglia and other tissue macrophages, conclu-sively establishing that microglial maintenancedid not rely on progeny of hematopoietic stemcells. With these insights came the ability toidentify microglial survival or maintenance fac-tors including colonystimulating factor (CSF)-1and interleukin (IL)-34 (Greteret al. 2012; Wanget al. 2012), both produced in the CNS by neu-rons during development and both ligands forthe CSF-1 receptor. Additionally, transcriptionfactors such as interferon regulatory factor(IRF)-8 could be shown essential for microglialphenotypes by Kierdorf and Prinz (2013).

In the following pages, we will review re-cent advances in our understanding of the roleof microglia in physiological and pathologi-cal processes of the CNS. Because of the promi-nent roles CX3CR1 and its ligand fractalkineplayed in bringing about these advances, andbecause fractalkine-induced, CX3CR1-mediat-ed signaling appears to regulate microglialbehavior in a number of CNS disorders in-cluding AD and MS, we will discuss the physio-logical roles of microglia as viewed from theCX3CR1–fractalkine perspective, providing aunique viewpoint. Based on the most recentstudies of molecular profiling of microglia, wewill also propose a molecularand functional def-inition of microglia that incorporates the prop-erties attributed to these cells in recent years.

PHYSIOLOGICAL ROLES OF MICROGLIAVIEWED FROM THE FRACTALKINE-CX3CR1PERSPECTIVE

Fractalkine (CX3CL1) and its receptor(CX3CR1) have assumed a prominent andunique role in CNS development, homeostasis,and disease within a relatively short time. Themost salient and well-established neurobiologi-cal role of this ligand/receptor pair is definedin large part by their cellular sources. Fractal-kine is made by neurons and the receptor isexpressed by microglia. Therefore, fractalkine/receptor interactions constitute a neuron–mi-croglial signaling system. Simply stated, effectsof neuronal fractalkine mediated by microglialfractalkine receptor provide a capsule view ofneuron–microglial cross talk throughout devel-opment, physiology, aging, and disease.

Early History of Fractalkine and Its Receptor

Fractalkine is a chemokine, albeit unusual. Che-mokines were identified by virtue of mediat-ing leukocyte subtype-specific chemotaxis invitro in 1987 (Ransohoff 2005). Four years lat-er, cloning of the first chemokine receptorcDNA (Horuk 1994) showed that chemokinereceptors belong to the superfamily of G pro-tein–coupled receptors (GPCRs), and subse-quent work showed that chemokine receptorscomprise a GPCR subclass signaling throughGai. Genomic analysis assigned most chemo-kines and chemokine receptors to a restrictedset of chromosomal loci, indicating their statusas a coherent genetic and functional family(Charo and Ransohoff 2006). At present, hu-mans and mice possess approximately 50 che-mokines and 20 chemokine receptors. Two largestructurally defined families, termed CC andCXC chemokines, include more than 90% ofall chemokines. In primary structures of CCchemokines (CCL[ligand]1–28), two position-ally conserved cysteines are side by side, whereasCXC (CXCL1–16) chemokines possess an in-tervening residue between the cysteines. CCchemokines signal almost without exceptiononly to CC chemokine receptors (CCR[recep-tor]1–12), and CXC chemokines show a similar


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preference for a corresponding set of CXC che-mokine receptors (CXCR1-7) (Rot and von An-drian 2004). Ligand–receptor relationships inthe chemokine universe are complex. Chemo-kine receptors can respond to as many as 10ligands, whereas individual chemokines cansignal to as many as three receptors.

Virtually all chemokines are small (10–14 kDa) secreted peptides, transcribed rapidlyand abundantly on demand with short-livedmessages and quickly cleared by degradationof protein and message as well as receptor-me-diated uptake and disposal of the peptides (Car-dona et al. 2008).

From their initial identification until 1998,chemokines were viewed mainly in the contextof inflammation and immunity because of theirpotent, selective action toward leukocyte popu-lations. Deletion of chemokine receptor CXCR4from the mouse genome upended this view ofchemokine physiology, showing prominent de-fects in multiple tissues, including abnormallocalization of numerous neuronal populations(Ma et al. 1998; Zou et al. 1998). These resultsushered in an era of chemokine neurobiology,with fractalkine constituting a conspicuous ele-ment.

Fractalkine was identified by molecularcloning in 1997 (Bazan et al. 1997) and showedtwo unusual features. First, the primary trans-lation product was not secreted and showed asingle-pass transmembrane region, long mucinstalk, and amino-terminal chemokine ectodo-main. Second, the canonical cysteine residuesshowed a CXXXC (CX3C) motif. In 2014, frac-talkine and fractalkine receptor remain the solemembers of their chemokine family. No otherCX3C chemokines have been discovered andonly one additional membrane tethered chemo-kine (CXCL16) has emerged. Fractalkine/frac-talkine receptor appear, for practical purposes,to constitute a monogamous ligand/receptorsystem. One other signaling CX3CR1 ligandwas reported (CCL26) but follow-up has beenscant so its biological significance is uncertain(Nakayama et al. 2010).

Fractalkine’s unusual name deserves a com-ment. Apparently the lead author was remindedof fractals, in which a repeating subunit recapit-

ulates the whole, by fractalkine’s primary struc-tural characteristics.

Fractalkine was quickly shown to be ex-pressed by endothelial cells, regulated in partby inflammatory cytokines. Initial studiesshowed that fractalkine signaled to receptor-bearing cells (mainly monocytes and naturalkiller [NK] cells) in two distinct ways. Eithermembrane fractalkine activated membrane frac-talkine receptor in a contact-dependent fash-ion, or the chemokine domain, released byproteolysis, could function as a soluble ligand.The transmembrane expression of fractalkineat endothelial apical surfaces suggested a meansof signaling to nearby leukocytes, and soon itwas shown that fractalkine/receptor interac-tions could arrest leukocytes under flow withoutinvolvement either of pertussis toxin-sensitiveG proteins or leukointegrins (Fong et al. 1998;Haskell et al. 1999, 2000).

Subsequent research showed that the solu-ble chemokine domain, mounted at the termi-nus of the mucin stalk, signaled by G protein–dependent interactions and mobilized calciumbut failed to activate integrins, a property incontrast to virtually all soluble chemokines.These experiments, performed nearly 20 yearsago, may be worth revisiting for guidance aswe seek to determine mechanisms by whichneuronal fractalkine signals to microglia, boththrough contact-dependent interactions and ata distance using soluble fractalkine. In additionto Gai-mediated reduction of cAMP, chemo-kine receptors signal to elevate cytosolic calci-um, activate mitogen-activated protein (MAP)kinases, and stimulate PI3K-Akt pathways. b-Arrestin recruitment and consequent receptorinternalization, at times with additional signal-ing outputs, also characterizes chemokine re-ceptors. These canonical, generic pathways pro-vide a platform for detailed characterization,which remains to be completed for practicallyall chemokine receptors in their native cellularcontexts. Only then will selective cellular re-sponses to chemokines become susceptible totherapeutic manipulation, beyond blockade oragonism.

For soluble fractalkine, structural studies in-dicate that its receptor-binding properties differ





substantially from those generally reported forchemokines (Mizoue et al. 1999), suggestingthat an account of fractalkine/receptor signal-ing may well be useful for translational ap-plications. Membrane fractalkine also showeddistinct properties, as substitution of other che-mokine domains at the end of the mucin stalkdid not produce chimeric molecules that couldsupport leukocyte arrest under flow (Haskellet al. 2000). Further, the fractalkine chemokinedomain’s crystal structure showed an unusu-ally compact globular domain, suggesting amechanistic explanation for the chemokine’sdistinct functional properties (Hoover et al.2000). Outcomes of soluble versus membranefractalkine signaling modes differ in relevantdisease models of neurodegeneration. Despiteextensive descriptive cell-biology investigationof membrane and soluble fractalkine, relativelylittle is known of differential downstream sig-naling cascades.

Fractalkine/Receptor in the Nervous System

Soon after its discovery, it became clear thatfractalkine might participate in neurobiologi-cal processes. Database inspection showed thata large majority of expressed sequence tags(ESTs) for fractalkine and its receptor camefrom CNS sources, providing a rough estimateof relative tissue expression levels. Elegant insitu hybridization (Harrison et al. 1998) showeda highly provocative expression pattern. Fractal-kine was produced by neurons, with the recep-tor restricted to microglia, and it was prescientlypredicted that neuron–microglial cross talkmight be supported by this cellular distributionpattern. Surprisingly, fractalkine’s expressionpattern throughout the remainder of the bodywas starkly different, being mainly on endothe-lium. Subsequent work confirmed that fractal-kine derives from neurons in the CNS and thatthe cerebral vasculature is an outlier in its clearlack of fractalkine expression (Sunnemark et al.2005).

Within a year, reports came suggesting thatfractalkine exerted unexpected effects towardmicroglia, especially considering that chemo-kines were largely deemed inflammatory ele-

ments, which orchestrated accumulation of im-mune cells and then promoted their effectorproperties. Instead, fractalkine protected mi-croglia from Fas-mediated cell death by alter-ing the phosphorylation of apoptotic regulatorssuch as Bcl-2 and Bad (Boehme et al. 2000).Others showed that neutralizing fractalkineantibodies nearly doubled microglial produc-tion of tumor necrosis factor (TNF) in responseto lipopolysaccharide (LPS) and exerted thesame magnitude of effect on microglial toxicitytoward cocultured hippocampal neurons. In animportant follow-up experiment (Zujovic etal. 2001), this group showed in vivo that intra-cerebroventricular (ICV) installation of fractal-kine antibodies greatly increased the inflamma-tory response to ICV TNF-a. Interestingly, co-injection of fractalkine along with TNF-a hadno effect at all, strongly suggesting that tonicsoluble fractalkine signaling was not a limit-ing factor for fractalkine-mediated neuropro-tection. The cumulative interpretation of thesestudies was that fractalkine tempered microglialresponses to cardinal inflammatory stimuli.Further, without the moderating effects exertedby fractalkine, the microglial reactions to thesestimuli could potentially be neurotoxic.

Other research implied greater complex-ity; in particular, neurons in vitro and in an invivo stroke model responded to excitotoxicstimuli by increased proteolytic release of frac-talkine (Chapman et al. 2000) with no changein mRNA level. In other cells, it was shown thatproteases of the A disintegrin and metallopro-tease (ADAM) family were responsible both fortonic (Hundhausen et al. 2003) and inducible(Garton et al. 2001; Tsou et al. 2001) fractalkinerelease. Because metalloproteases were also im-plicated in fractalkine release from neurons(Chapman et al. 2000), it seems plausible thatthese same factors mediate its proteolysis fromsome neuronal cells in hippocampus and cor-tex. Subsequent work showed that cathepsin S,a distinct protease (Clark et al. 2009), was re-quired to generate soluble fractalkine from ratdorsal horn neurons.

Fractalkine signaling was also implicated inindirect neuroprotection. One particularly lu-cid series of experiments showed that neuronal





fractalkine elicited adenosine release from mi-croglia, activating survival pathways in excito-toxicity-challenged neurons via adenosine A1receptors (Lauro et al. 2008, 2010). Initial find-ings in vitro were extended in vivo to a perma-nent focal ischemia model (Cipriani et al. 2011)and were enriched by showing that fractalkinesignaling also supported glutamate uptake byastrocytes (Catalano et al. 2013).

New In Vivo Tools Bring New Insights

In 2000 came the report of a genetic model forfractalkine receptor, in which the CX3CR1 cod-ing region had been replaced with GFP usinghomologous recombination, yielding mice inwhich cells transcribing the fractalkine recep-tor gene expressed GFP in the heterozygous(GFP/þ) or homozygous knockout (GFP/GFP) state (Jung et al. 2000). These mice showedno immediately evident phenotype in eitherCNS or peripheral immune responses (Junget al. 2000). As one example, the brain-stem mi-croglial morphological response to peripheralfacial nerve crush, an established model for in-ducing microgliosis (Graeber et al. 1998), wasnot affected in fractalkine receptor knockoutmice. Results were confirmed in other geneticmodels of fractalkine receptor deficiency (Has-kell et al. 2001; Tripp et al. 2001).

At about the same time, mice lacking frac-talkine were generated (Cook et al. 2001; Sor-iano et al. 2002), enabling investigators to ask ifligand and receptor-knockout mice would showcompatible phenotypes, an essential confirma-tion step for studies using these monogamousligand/receptor genetic models.

CX3CR1gfp mice showed distinct advantag-es for monitoring microglial dynamics in vivousing two-photon imaging. As described above,these mice were used to establish the remark-ably ceaseless movements of microglial process-es (Davalos et al. 2005; Nimmerjahn et al. 2005)along with their lightning-like responses to mi-croscopic foci of injury (Davalos et al. 2005).Thereafter, the concept of “resting microglia”became progressively obsolete. In initial exper-iments, mice deficient for fractalkine showedsmaller stroke volume after transient focal is-

chemia (Soriano et al. 2002), suggesting thatfractalkine/fractalkine receptor inflammatorysignaling worsened tissue damage.

Fractalkine Receptor Signaling IlluminatesRoles of Microglia in Neurodevelopment

It has become clear that parenchymal microgliaenter the CNS parenchyma early (�E10.5 inmice) during development (Ransohoff andCardona 2010) and that their proliferation andmaintenance depend in part on factors elabo-rated from neurons. After seminal documenta-tion (Tremblay et al. 2010; Schafer et al. 2012)that microglia mediate complement-dependentsynaptic pruning in some regions of the CNS, itwas pertinent to ask whether fractalkine signal-ing contributed to regulating this process, asfractalkine receptor was shown present on mi-croglial progenitors from their first entry intoparenchyma (Mizutani et al. 2012). Results ofexamining synaptic maturation in the hippo-campus of fractalkine receptor-deficient ani-mals indicated a substantial delay in CX3CR1knockouts, potentially caused by transient re-duction in microglial numbers (Paolicelli et al.2011; Ransohoff and Stevens 2011). Further,CX3CR1-deficient mice showed severely alteredsocial interaction coupled to defective networkinteractions in adulthood (Paolicelli and Gross2011; Zhan et al. 2014).

By analogy with macrophages, microgliamay carry out two major functions during de-velopment: phagocytosis and trophic-factorproduction. Phagocytosis is exemplified by syn-aptic pruning. Factor production also occursduring development and is pivotal for certainneuronal populations. Layer V cortical neuronsshow decreased survival during the first postna-tal week in fractalkine receptor deficient mice,because of reduced microglial production ofinsulin-like growth factor (IGF)-1 (Ueno et al.2013).

Microglia Play a Role in Learning-DependentSynapse Remodeling

With advancement in Cre-Lox technologies, itwas only a matter of time before the properties





of CX3CR1 as a receptor expressed uniquely onmicroglia were used to target microglia and/orspecific genes in microglia to understand theirphysiological function. Using CX3CR1 CreERmice expressing tamoxifen-inducible Cre re-combinase that allow for specific expressionof the diphtheria toxin receptor in microglia,Parkhurst et al. (2013) were able to specifical-ly deplete microglia from the brain followingdiphtheria toxin administration. Mice depletedof microglia showed deficits in multiple learn-ing tasks and a significant reduction in motor-learning-dependent synapse formation. Moreinteresting is that Cre-dependent removal ofbrain-derived neurotrophic factor (BDNF) frommicroglia recapitulated one salient effect of mi-croglia depletion: impaired motor plasticity.Further, microglial BDNF increased neuronaltropomyosin-related kinase receptor B phos-phorylation, a key mediator of synaptic plastic-ity. These findings suggest that microglia mayhave important physiological functions in learn-ing and memory by promoting learning-relatedsynapse formation through BDNF signaling.A detailed discussion of the role of fractalkine-CX3CR1 signaling in synapse development andpruning can be found in Schafer and Stevens(2015).

MICROGLIA IN CNS DISORDERS

Significant advances have been made in under-standing the roles of microglia in normal brainfunctioning. In contrast, understanding the roleof microglia in CNS disorders has proven moredifficult. Brain pathology is frequently associ-ated with disruption of the blood–brain bar-rier (BBB) and recruitment of peripheral bloodmonocytes. These cells, although significantlydifferent from microglia, share several function-al and transcriptional characteristics with mi-croglia, making it more difficult to dissect theirroles from those of microglia. However, the newemerging technologies described above, whichallow specific targeting of microglial genes,could make such a task slightly easier. We willfocus here on MS and AD, two major disordersof the CNS.

Microglia in MS

It is quite possible that the first description ofmicroglia in the brain and spinal cord of a pa-tient with MS was made by Carl Froininann whoshowed that cells (likely microglia) changedtheir morphology in CNS tissues from a 22-yr-old MS patient (Froininann 1878). However,despite of decades of research, the exact role ofmicroglia in MS has not been clarified. In ani-mal models of MS, macrophages and microgliahave been reported to exert detrimental func-tions, including toxicity to neurons and oligo-dendrocyte precursor cells, release of proteases,production of inflammatory cytokines and re-active oxygen and nitrogen species, and recruit-ment and reactivation of T lymphocytes. Manystudies, however, have also reported beneficialroles of macrophages and microglia, includ-ing roles in axonal regeneration, promotion ofremyelination, clearance of inhibitory myelindebris, and the release of neurotrophic fac-tors (Rawji and Yong 2013). A main point ofconfusion is that investigators have used mi-croglia and macrophages interchangeably. Add-ing to the conceptual chaos, in EAE, a model forthe inflammatory aspects of MS (Huang et al.2006), fractalkine/receptor interactions medi-ate recruitment of disease-suppressing NK cells(Huang et al. 2006), which was shown subse-quently to be a dominant effect (Garcia et al.2013) over the effects of fractalkine/receptorinteractions on microglia. The fog may be be-ginning to dissipate, however, on the role ofmicroglia in EAE. Recently, using a CX3CR1-Cre mouse model similar to the one describedby Parkhurst et al. (2013) above, Goldmannand colleagues (2013) found that conditionaldeletion of the transforming growth factor(TGF)-b-activated kinase 1 (TAK-1) in mi-croglia only, not in neuroectodermal cells, sup-pressed EAE disease, significantly reduced CNSinflammation and diminished axonal and mye-lin damage by cell-autonomous inhibition ofthe NF-kB, JNK, and ERK-1/2 pathways. Theseresults strongly suggest that microglia may pro-mote tissue injury in EAE. Some caveats applyhowever. It is not entirely certain when theTAK-1 deletion from microglia exerts its effects.





Further, it remains conceivable that the tran-sient peripheral deletion of TAK-1 from allCX3CR1þ cells (an eccentricity of this geneticmodel) may play some role in the findings.

Another set of informative data came fromexamining dual-knockin mice in which Ccr2was disrupted by insertion of red fluorescentprotein (RFP), and the cross of these mice withCX3CR1gfp animals yielded animals in whichboth Ly6Chi and Ly6clo monocytes were labeled,as well as the microglia (Fig. 1). Studies at EAEonset revealed that macrophages derived fromLy6Chi monocytes initiated demyelination andmade inflammatory mediators. In contrast, mi-croglia did not associate with undisrupted axo-glial units and showed an expression profile ofrepressed metabolism (Yamasaki et al. 2014). Itwill be useful to integrate the finding from thisstudy with those in which TAK-1 was deletedfrom microglia. It is reasonable to speculate thatmicroglial activities, which promote EAE, takeplace before disease onset.

Finally, of course, the relevance of these ob-servations to MS remains to be seen.

Microglia in AD

There is definitive evidence that AD is associ-ated with significant accumulation of microgliain senile plaques (Fig. 2). Work over the pastdecade, however, suggests that, in addition tomicroglia, other mononuclear phagocytes, suchas perivascular macrophages and circulatingmonocytes, may also be involved.

Microglia are two- to fivefold more concen-trated around plaques in AD patients and mousemodels of this disease (Frautschy et al. 1998)and express major histocompatibility complexII and markers of inflammation (Tooyama etal. 1990; McGeer and McGeer 2010). b-Amy-loid (Ab) activates neonatal mouse microgliain vitro to produce reactive nitrogen speciesand TNF-a (Meda et al. 1995), a process thatis mediated in part by binding Ab to a CD36-TLR4-TLR6 receptor complex on microglia (ElKhoury et al. 2003; Stewart et al. 2010). Ab alsoactivates the NLRP3 inflammasome in microg-lia leading to neurodegeneration (Heneka et al.2013).

Although we have some insight into howmicroglia change following interaction withAb, understanding how microglia accumulateat sites of Ab deposition has proven to bemore difficult, and hinted at a role for peripheralmonocytes in AD. Transgenic mice expressingthe human Ab protein precursor with the Swe-

Figure 1. Microglia begin to clear myelin debris atEAE onset. Three-dimensional reconstructionsfrom serial block face scanning EM of spinal cordtissues of a mouse at day of EAE onset show a largeflap of myelin debris (blue) engaged by a microglialprocess (green). A nearby monocyte (red cytosol;golden nucleus) enwraps a myelin internode and isactively removing myelin. Both monocyte and mi-croglial cell (with yellow nucleus) contain myelin in-clusions (blue), although those of the monocyte arelarger and more numerous.





dish mutation (APP Tg2576) and deficient in thechemokine receptor CCR2 (APP-CCR22/2)have reduced numbers of monocytes in thebrain early in the disease process, before forma-tion of senile plaques. This reduction in thenumber of monocytes was associated with in-creased mortality and higher Ab levels exclu-sively around blood vessels in the brain, suggest-ing that early monocyte accumulation promotesthe clearance of perivascular Ab and protectsmice from Ab toxicity early in the disease pro-cess (El Khoury et al. 2007). The process of Abclearance is at least, in part, mediated by phago-cytosis via the scavenger receptor SCARA1 (ElKhoury et al. 1996; Frenkel et al. 2013).

Perivascular Ab deposits are found in a highpercentage of AD patients and are a hallmark ofcerebral amyloid angiopathy. Because microgliaexpress little CCR2 (Mizutani et al. 2012), thesefindings raise the possibility that accumulationof microglia at parenchymal sites of Ab depo-sition involves a separate process that does notrequire CCR2, unlike what happens at perivas-cular sites of Ab deposition. This is indirectlysupported by results showing that selective de-pletion of perivascular macrophages exacer-bates perivascular Ab deposition, and stimula-tion of their turnover reduces perivascular load(Hawkes and McLaurin 2009). The distinctionbecomes less critical if, as proposed by Kumar-

Singh and colleagues (2005), all Ab depositsdevelop initially around blood vessels and thenthese vessels are obliterated leading to formationof the classic plaques. The latter possibility issupported by experiments in which adoptivelytransferred monocytes home to senile plaquesin a mouse model of AD (Lebson et al. 2010).

Regardless of whether Ab clearance isthe task of microglia or recruited perivascularmonocytes, the ability of microglia to clear Abappears to be reduced with disease progression.Expression of microglial Ab phagocytic recep-tors and Ab-degrading enzymes in a mousemodel of amyloid deposition in AD is signifi-cantly reduced (Hickman et al. 2008), suggest-ing that accumulation of Ab is in part the resultof a failure of microglia to adequately clear thistoxic peptide.

Additional insight into the role of microgliain AD came from analyzing the role of CX3CR1in mouse models of amyloid deposition andtauopathy. CX3CR1-deficient animals show en-hanced Ab clearance (Lee et al. 2010; Liu et al.2010), with a prominent gene-dosage effect. In-creased IL-1b is also present in the CNS of thefractalkine receptor knockout animals, and thephenotype resembles that of mice overexpress-ing IL-1b (Shaftel et al. 2007). In mice withtau pathology, absence of CX3CR1 substantiallyworsens disease progression, neuropathologi-cally, biochemically, and behaviorally (Bhaskaret al. 2010). It is plausible that increased mi-croglial production of IL-1b is responsibleboth for enhanced Ab clearance and for wors-ened tau pathology (Prinz et al. 2011).

Such findings raise the interesting ques-tion of whether soluble fractalkine (sFKN) andmembrane-tethered fractalkine (mFKN) arefunctionally distinct. The corollary questionis whether contact-dependent interactions be-tween neurons and microglia via fractalkine/re-ceptor interactions support the same functionsas does signaling at a distance through cleavedfractalkine. Studies in the periphery suggesteddistinct functions of the two-fractalkine iso-forms, as mFKN rescued survival of Ly6Clo

monocytes, whereas sFKN reversed a morpho-logical defect in CX3CR1þ gut dendritic cells(Kim et al. 2011). Use of recombinant adeno-

Figure 2. b-Amyloid (Ab) deposits are surroundedby microglia. Microglia, stained here in brown forCD11b, cluster around sites of Ab deposition, stainedin green with thioflavin S, in a mouse model of cere-bral amyloid deposition.





associated virus (AAV) vectors to deliver fractal-kine isoforms showed that sFKN (but notmFKN) reversed the severely augmented toxic-ity of MPTP for substantia nigra dopamine neu-rons (Morganti et al. 2012). This observationwas extended to a therapeutic paradigm by us-ing these AAV vectors to overexpress sFKN in afractalkine-sufficient model of genetic tau pa-thology (Nash et al. 2013), with substantial ben-efit. However, the same approach was neitherbeneficial nor deleterious in an amyloid depo-sition model (Nash et al. 2013), compatible withearlier results using the LPS challenge (Zujovicet al. 2001).

Because recent findings suggest that tau pa-thology may occur following Ab deposition,and based on the above discussion, we proposethat the role of microglia in AD evolves withdisease progression. Microglia initially interactwith Ab possibly in an attempt to clear it. Butthe continuous accumulation of Ab also leads toinduction of inflammatory cytokines that re-duce the Ab clearance ability of microglia andlead to tau pathology, initiating a self-perpetu-ating loop that culminates in worsening disease(see Table 1). This is reminiscent of the sequenceof events that occur in atherosclerosis, anotherdegenerative disease of aging. Monocytes areinitially recruited to sites of deposition of mod-ified lipoproteins, likely in an attempt to clearthem. Continued accumulation of such lipo-proteins overwhelms the ability of the mono-cytes to clear them and transforms these cellsinto foam cells that contribute to disease prog-ression. Of interest, scavenger receptors thatappear to mediate the interactions with Ab in

AD also mediate the interactions of monocyteswith modified lipoproteins in atherosclerosis.

In addition to their roles in MS and AD,microglia have been implicated in a numberof neurological and neuropsychiatric disorders(El Khoury 2010). For example, microglia ina mouse model of amyotrophic lateral sclerosis(ALS) have a distinct molecular signature (Chiuet al. 2013). A defect in the microglial/myeloidgene Hoxb8 is associated with compulsivegrooming similar to behavior in humans withobsessive–compulsive disorder (Chen et al.2010). Another example is Rett syndrome, anX-linked autism spectrum disorder. In a mousemodel of Rett syndrome, normal microgliacan arrest the pathology and attenuate diseasesymptoms (Derecki et al. 2012).

TOWARD A MOLECULAR DEFINITIONOF MICROGLIA

It is evident from the above discussion thatthe ability to distinguish microglia from othercells of the CNS as well as from other mononu-clear phagocytes is crucial to understanding thefunctions of these cells in physiological andpathological processes. Development of meth-ods to isolate microglia from adult animals withhigh purity and yield as well as recent advancesin approaches to define the transcriptomes ofvarious cells by RNASeq and microarrays al-lowed four independent groups to define thetranscriptomes of adult mouse microglia andmake these data publicly available in 2013(Beutner et al. 2013; Chiu et al. 2013; Hickmanet al. 2013; Butovsky et al. 2014). These resultsshowed that, although microglia share severalimportant transcripts with other mononuclearphagocytes, they also have significant differenc-es. Analysis of these datasets shows that P2ry12and P2ry13, Tmem119, Gpr34, Siglech, Trem2,and CX3CR1 are among the transcripts unique-ly expressed in microglia compared with oth-er mononuclear phagocytes. Macrophage-en-riched genes include fibronectin, the chemokineCxcl13, and the endothelin B receptor.

Analysis of these datasets also allowedcorrelation between microglial gene expressionand function. For example, a cluster of genes

Table 1. The neuroimmune system plays beneficialand detrimental roles in Alzheimer’s disease

Potential beneficial roles

Potential detrimental

roles

Ab phagocytosisRelease of Ab-degrading

enzymesClearance of debris and

degenerating neuronsRelease of neurotropic

factors

Release of inflammatorycytokines

Release of reactiveoxygen and nitrogenspecies





termed “sensome” was introduced that definesthe genes used by microglial to sense their en-vironment (Hickman et al. 2013), a major func-tion of microglia described using two-photonimaging (Davalos et al. 2005; Nimmerjahn et al.2005). This cluster includes 100 genes that con-stitute the microglial toolset for sensing changesin the brain’s milieu and define the apparatusthat microglia use to perform various homeo-static functions including sensing of chemo-kines and cytokines, purinergic molecules, in-organic substances, changes in pH and aminoacids. Defining the microglial sensome underphysiological conditions, establishes a baselineto which we can compare and identify changesthat occur in this sensome under pathologi-cal conditions. Highlighting the importance ofthese datasets, two members of the microglialsensome, TREM2 and CD33, have been foundto be independent risk factors for late onset AD(Jonsson et al. 2012; Bradshaw et al. 2013; Guer-reiro et al. 2013).

Another important result in these datasetsis the finding that resident microglia expresshigh levels of several antimicrobial peptidesnot previously known to be expressed on thesecells (Hickman et al. 2013). These includeCamp, the cathelicidin-related antimicrobialpeptide and Ngp, the neutrophilic granule pro-tein. The high level of expression of these pep-tides indicates a high level of readiness by thenormal “quiescent” resident microglia to per-form its innate host defense function in the ab-sence of adaptive immune molecules and cells.These findings further support the concept thatthere is no “resting” microglia, and that thesecells are highly dynamic cells important for sur-veillance, protection, and housekeeping of theneural milieu.

CONCLUSIONS

It is an exciting time to study microglia. Basedon the rapid pace of discovery in the past de-cade, and the continued development of newtechnologies, it is highly likely that the nextdecade will bring our level of understandingof these cells on par to our levels of understand-ing of other innate immune cells in the periph-

ery and allow us to harness such knowledge todefine the role of these cells in normal and path-ological processes and to possibly target thesecells for diagnosis and/or treatment of variousCNS disorders. To fully define the exact roles ofmicroglia in these disorders, experiments map-ping the gene expression profiles of microgliaand other neuroimmune cells in mouse modelsand patient samples and at multiple stages ofvarious CNS disorders need to be performed(El Khoury 2010).

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September 9, 20152016; doi: 10.1101/cshperspect.a020560 originally published onlineCold Spring Harb Perspect Biol

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